Calcium dysregulation combined with mitochondrial failure and electrophysiological maturity converge in Parkinson’s iPSC-dopamine neurons

Summary Parkinson’s disease (PD) is characterized by a progressive deterioration of motor and cognitive functions. Although death of dopamine neurons is the hallmark pathology of PD, this is a late-stage disease process preceded by neuronal dysfunction. Here we describe early physiological perturbations in patient-derived induced pluripotent stem cell (iPSC)-dopamine neurons carrying the GBA-N370S mutation, a strong genetic risk factor for PD. GBA-N370S iPSC-dopamine neurons show an early and persistent calcium dysregulation notably at the mitochondria, followed by reduced mitochondrial membrane potential and oxygen consumption rate, indicating mitochondrial failure. With increased neuronal maturity, we observed decreased synaptic function in PD iPSC-dopamine neurons, consistent with the requirement for ATP and calcium to support the increase in electrophysiological activity over time. Our work demonstrates that calcium dyshomeostasis and mitochondrial failure impair the higher electrophysiological activity of mature neurons and may underlie the vulnerability of dopamine neurons in PD.


INTRODUCTION
Mutations in the b-glucocerebrosidase (GCase) enzyme encoded by the GBA gene are the strongest common genetic risk factors for late onset Parkinson's disease (PD). The GCase enzyme metabolizes glucocerebroside into glucose and ceramide within lysosomes. 1 Cells carrying heterozygous GBA mutations such as N370S and L444P exhibit approximately 50% of wild-type GCase activity, increased GCase protein misfolding and endoplasmic reticulum (ER) stress which contribute to the risk of developing PD likely through a combination of loss and gain of function. [2][3][4] Although the reduction in GCase activity impacts the autophagic/lysosomal pathway implicated in PD, evidence also supports the impact of GCase mutations on other neuronal functions. These include neurotransmitter release, neuronal ultrastructure, a-synuclein neuronal accumulation and release, and calcium handling, pointing toward a more complex role of GBA in PD pathology. [4][5][6] Calcium is essential in a number of critical neuronal functions, such as neurotransmitter release, pacemaker activity and neuronal excitability, [7][8][9][10][11] with calcium dysregulation well-described as a major cellular phenotype in PD. 12,13 Calcium is also essential for ATP generation, 14 important for neuronal functions such as ion channel regulation, neurotransmitter vesicle release/reuptake and synaptic receptor activation. [15][16][17][18] Calcium and ATP are, therefore, at the center of the neuronal bioenergetic pathways which may underpin the preferential vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNpc) in PD. SNpc neurons possess long unmyelinated arborized processes and display an order of magnitude more synapses (approximately between 1 and 2.5 x 10 6 -fold more release sites) than other neuronal subtypes 19 conveying a high metabolic demand.
Calcium dysregulation is evident in human iPSC-derived neuronal models of PD. The GBA-L444P mutation induces dysfunctional organelle calcium handling in neurons elevating both basal levels of somatic calcium and release of calcium from the ER, compared to controls. 6 We have previously shown that GBA-N370S iPSC-derived dopaminergic neuronal cultures have significantly elevated levels of calcium-regulating

Electrophysiological maturity of iPSC-dopamine neurons progresses over time
Previous work [24][25][26][27][28] has shown that although iPSC-derived neurons display neuronal subtype-specific molecular markers at relatively early timepoints, synaptic function and neurotransmission take longer to evolve. 24,[29][30][31] To ascertain when iPSC-derived dopamine neurons display the electrophysiological activity critical to their physiological role, we performed whole cell patch clamp characterization on control iPSCdopamine neurons generated using a previously described protocol 28 to produce tyrosine hydroxylase (TH)-positive neurons ( Figure S1). Electrophysiological measurements were obtained at several timepoints between 35 and 100+ days in vitro (DIV), and the data placed into 10-day blocks. In this manner, we ascertained the development of passive properties including an increase in cell capacitance ( Figure 1A) and a concurrent decrease in membrane resistance ( Figure 1A), over time. These likely represent an increase in neuronal arborization and insertion of functional ion channels into the membrane, respectively. Resting membrane potential (RMP) became more hyperpolarized over time ( Figure 1B) until close to the resting membrane potential of a dopamine neuron in vivo (approximately between À40 and À57 mV 32,33 ). Notably, an increase in staining of pre-and post-synaptic markers (Synapsin-1 and Homer, respectively) in apposition ( Figure 1C), was observed, coincident with a dramatic increase in spontaneous excitatory postsynaptic currents (sEPSC; Figure 1D), confirming the formation of fully functional synapses at approximately 70 DIV. Furthermore, intrinsic pacemaking activity insensitive to inhibitors of network activity (CNQX, AP5 and iScience Article bicuculine; Figure S2) was also seen at $70 DIV ( Figure 1E). These features of fully functional dopaminergic neurons were accompanied by the presence of large A-type K v 34,35 and hyperpolarization-induced membrane potential sag [36][37][38] ( Figure S2) demonstrating that these neurons could be considered electrophysiologically mature by 70 DIV. We therefore chose to use 35 DIV and 70+ DIV as timepoints to compare biological phenotypes at early and late stages of neuronal maturity, respectively.

GBA-N370S iPSC-dopamine neurons display reduced mitochondrial calcium signaling
Acute intracellular calcium release from organelle stores such as mitochondria and endoplasmic reticulum (ER) is essential for neuronal functions such as synaptic plasticity and neurotransmitter release. 39 We have previously shown that calreticulin, a protein important in calcium homeostasis in intracellular stores, was elevated in PD GBA-N370S iPSC-dopamine neurons. 4 To investigate calcium signaling and release, control and GBA-N370S iPSC-dopamine neurons were assessed for calcium dysregulation using ratiometric calcium recordings. Using acute ionomycin stimulation known to result in organellar calcium release, 40,41 we compared iPSC-dopamine neurons generated from healthy control iPSC lines, and from iPSC lines carrying the GBA-N370S, across multiple different differentiations. Mature neurons at 70+DIV of control but not GBA-N370S genotypes had a significant increase in somatic calcium concentration after ionomycin stimulation as detected using the ratiometric dye Fura-2. When the difference between baseline and maximal store release (at approx. 6-10 s post-stimulation) was compared we found that GBA-N370S iPSC-dopamine neurons released significantly less calcium than controls ($50% reduction; p = 0.0344; Figure 2A). We then showed that this effect was present at earlier stages of neuronal development (35 DIV; Figure S3).  (H) GBA-N370S iPSC-dopamine neurons exhibit reduced ER/mitochondrial interaction by proximity ligation assay (PLA) puncta. Lines used: Ctrl 2,3,4,5&8 and GBA-N370S 1,3,4,5 over 2 differentiations. Left: example image for ER-Mitochondria-PLA. Red illustrates IP3R3 (ER) and VDAC1 (mitochondria) interaction; Green illustrates tyrosine hydroxylase and blue illustrates DAPI stain (scale bar = 50mm). Right: Average PLA puncta per TH positive neuron relative to control. Adjoining lines represent a differentiation (p = 0.000353 using random intercepts model). All data represented as mean G SEM. All significances are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

OPEN ACCESS
iScience 26, 107044, July 21, 2023 5 iScience Article As ionomycin works on multiple intracellular calcium stores, including the ER and mitochondria but not lysosomes, 42 we sought to identify which store was responsible for the release deficit to understand the impact of aberrant calcium homeostasis on neuronal dysfunction. We first demonstrated using our Fura-2 assay for cytoplasmic calcium that ER calcium leak did not differ between genotypes after inhibiting the SERCA pump with cyclopiazonic acid (CPA) ( Figure 2B). To confirm this finding, we then investigated ER calcium leak using the genetically encoded ER calcium sensor GCEPIAer, 43 a method which allows calcium dynamics to be measured specifically from the ER. Using the GCEPIAer sensor we found no significant differences in ER calcium leak between iPSC-dopamine neurons derived from individuals with the GBA-N370S mutation and those from controls after CPA treatment ( Figure 2C). Finally, depolarization of the mitochondrial membrane potential using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showed significantly decreased calcium release from the mitochondria in iPSC-dopamine neurons derived from individuals with the GBA-N370S mutation compared to controls ( Figure 2D).
PLD1 and iPLA2 link aberrant mitochondrial calcium signaling and intracellular stores replenishment in GBA-N370S iPSC-dopamine neurons Next, we identified potential mechanisms which may underlie the dysregulation of calcium homeostasis. We found that phospholipase D1 (PLD1; an important neuronal calcium signaling molecule 44 ) was significantly decreased by 50% in GBA-N370S iPSC-dopamine neurons at the protein level ( Figure 2E). To validate the involvement of PLD1 in calcium dysregulation we used CRISPR technology to knockdown PLD1 in the SH-SY5Y neuroblastoma cell line. Lentiviral transduction of gRNAs reduced PLD1 expression by 50% in SH-SY5Y cells ( Figure 2E). Ionomycin stimulation of PLD1-knockdown (KD) cells resulted in a significant reduction in the amount of calcium released from intracellular stores into the cytoplasm as detected by Fura-2 compared to parental control cells ( Figure 2F), phenocopying the calcium phenotype observed in GBA-N370S iPSC-dopamine neurons.
In addition to a reduction in the calcium release signaling protein PLD1, we observed a reduction in expression of calcium independent phospholipase 2 (iPLA2) protein in GBA-N370S iPSC-dopamine neurons ( Figure 2G). The iPLA2 protein is important in replacing depleted organelle calcium stores via store operated calcium entry. Mutations in iPLA2 (PARK14) are associated with early-onset PD 45,46 and lead to reduced replenishment of organelle calcium stores. 47 The reduction in PLD1 and iPLA2 levels in GBA-N370S at 35 DIV iPSC-dopamine neurons suggests the neurons have deficits in both calcium release and calcium replenishment to stores.
In addition to disrupted signaling and replenishment of stores, the mitochondrial calcium deficit could result from decreased calcium flux from ER stores. The ER is the primary source of mitochondrial calcium so decreased ER-mitochondrial proximity would result in a reduction in mitochondrial calcium. Using the Proximity Ligation Assay with markers for mitochondria (VDAC1) and ER (IP3R3) we showed that there was a significant decrease in ER-mitochondrial proximity in neurons derived from GBA-N370S lines compared to controls ( Figure 2H).

GBA-N370S iPSC-dopamine neurons exhibit perturbed mitochondrial function and mitochondrial stress
Decreased calcium release from mitochondrial stores may reflect decreased mitochondrial calcium content, leading to decreased mitochondrial dehydrogenase enzymatic activity and mitochondrial dysfunction. 14, 48 To test whether GBA-N370S iPSC-dopamine neurons have dysfunctional mitochondria, we measured oxygen consumption rate (OCR) of basal, maximal, spare and ATP-associated respiration in control and GBA-N370S iPSC-dopamine neurons at the early (35 DIV) and mature (70+ DIV) time points using the Seahorse flux analyzer which measures the oxygen content in the media surrounding cells to assay oxygen consumption because of mitochondrial respiration. We observed decreased OCR of basal and maximal mitochondrial function in the GBA-N370S iPSC-dopamine neurons compared to controls at 35 DIV ( Figures 3A and 3B). In addition, the spare capacity of GBA-N370S iPSC-dopamine neurons was significantly reduced ( Figure 3B). As predicted, the amount of OCR attributed to ATP production was also significantly lower, meaning less ATP is generated by GBA-N370S iPSC-dopamine neurons per oxygen molecule ( Figure 3B). This effect persisted over time and the OCR attributed to ATP generation in GBA-N370S iPSCdopamine neurons remained significantly lower at the later timepoint (70+ DIV; Figure 3B) with basal, maximal and spare capacity OCR also significantly reduced. OCR values (basal, maximal, ATP and spare capacity) were markedly increased in both genotypes over time (i.e. between 35 DIV to 70+ DIV; data not shown), another indication of increasing maturity over time.

OPEN ACCESS
The reduced generation of ATP by mitochondria in GBA-N370S iPSC-dopamine neurons may reflect an inability to meet the bioenergetic demand as a result of decreased mitochondrial calcium, as opposed to a decreased ATP requirement of the neuron. Mitochondrial membrane potential (Dcm) drives ATP generation, with chronic changes to Dcm also indicating mitochondrial stress. To investigate this possible mechanism for dysfunctional mitochondrial OCR we measured Dcm using JC-10, a ratiometric dye which enters mitochondria and is used calculate mitochondrial membrane potential ( Figure 3C). We observed decreased mitochondrial membrane potential in GBA-N370S iPSC-dopamine neurons at 35 DIV (p = 0.00221; Figure 3Di) and at 70+ DIV (p = 0.00759; Figure 3Dii), confirming the compromised mitochondrial function in PD neurons.
Spontaneous quantal neurotransmitter release is compromised and synaptotagmin-1 levels are reduced in GBA-N370S iPSC-dopamine neurons Taken together, these data support a hypothesis of unbalanced energy supply and demand as GBA-N370S iPSC-dopamine neurons mature. The increasing bioenergetic demand imposed by a rising, high frequency and persistent electrophysiological activity is likely unsustainable in the face of low levels of mitochondrial calcium and ATP production and will either result in a reduction of neuronal activity, or cell death.
To investigate this further, we compared electrophysiological recordings of GBA-N370S and healthy control iPSC-dopamine neurons. At 70+ DIV, when heightened spontaneous activity was observed in healthy neurons (Figure 1), we showed that the frequency of spontaneous excitatory postsynaptic currents (sEPSC) which are dependent on calcium and ATP 16,49 were significantly reduced in GBA-N370S iPSC-dopamine neurons ( Figure 4A). In contrast, the pacemaking activity characteristic of SNpc neurons was unchanged in GBA-N370S iPSC-dopamine neurons ( Figure 4B). Induced action potentials (APs) showed no significant difference in frequency (with increasing input above rheobase) or duration, implying that the GBA-N370S iPSC-dopamine neurons are capable of heightened activity if forced ( Figure 4C). Current density from voltage gated channels showed no significant difference between GBA-N370S and control iPSC-dopamine neurons ( Figure 4D), also implying that GBA-N370S iPSC-dopamine neurons are capable of generating the same number of APs and action potential duration as control neurons if stimulated.
Calcium is responsible for regulation of neurotransmitter release and electrophysiological activity in neurons, 50 a process controlled in part by specialized calcium binding proteins such as synaptotagmin-1. Synaptotagmin-1 tethers to and relocalizes synaptic vesicles based on their calcium bound state 51 and is implicated in fast dopamine transmission. We found the levels of synaptotagmin-1 protein to be significantly reduced in whole cell lysates of GBA-N370S at 70+ DIV in iPSC-derived dopamine neurons which may explain the reduced neurotransmitter release and sEPSC frequency compared to controls ( Figure 4E).
Taken together, these data show the reduction of the key calcium regulatory proteins PLD1, iPLA2 and synaptotagmin-1 in GBA-N370S iPSC-dopamine neurons suggest a possible link between mitochondrial calcium dysregulation, reduced mitochondrial function, and impaired neurophysiological activity which may ultimately contribute to the preferential vulnerability of dopamine neurons in PD.

DISCUSSION
It is essential to understand the order of appearance of pathophysiological phenotypes in neurodegeneration to develop disease modifying interventions effective in an early therapeutic window. Here we (D) Delta fluorescent ratio from the JC10 assay measuring mitochondrial membrane potential. Each pair represents the average ratio normalized to average control for each differentiation (Lines used: Ctrl 1-6 and GBA 1-5). All data represented as mean G SEM. All significances are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using a random intercepts model. iScience Article describe a pathophysiological timeline in which calcium release and store content deficits in mitochondria cause mitochondrial stress and a reduced ability to produce ATP. The resulting decrease in calcium and ATP leads to decreased spontaneous neuronal activity (measured by sEPSC) which ultimately compromise neuronal function and contributes to neuronal vulnerability.
In recent years, changes in electrophysiological activity as an early disease marker have been the focus of investigation within the PD field. 52,53 Here we observed a decreased spontaneous electrophysiological activity after 70 days of neuronal maturation, a phenotype not previously described in a GBA-PD model. Electrophysiological dysfunction and aberrant activity have been described in both PD mouse models and human patients, occurring before the development of motor phenotypes, but coincident with cognitive perturbations. [54][55][56] These data support the concept that neuronal dysfunction precedes degeneration in disease progression. Indeed, alterations to excitability before neuronal death is seen in other neurodegenerative disorders, for instance in Huntington's disease and amyotrophic lateral sclerosis. 57,58 The previously-undescribed synaptic phenotype we present here is consistent with the growing concept that perturbations in neuronal activity represent an early aspect of neurodegeneration.
The decreased activity we observe in GBA-N370S iPSC-dopamine neurons could represent a normal neuronal plastic adaptation in response to persistent decreased calcium levels. This would likely result in alterations in ion channel expression or synapse number, changing the probability of release (P r ). The function of unitary sEPSC is still unclear but is distinct from action potential generated neurotransmitter release 59 and has been linked with synaptic development. 60 The reduced activity displayed in GBA-N370S iPSC-dopamine neurons may therefore cause an alteration in neuronal development which may play a role in early stages of genetic forms of PD.
PLD1 is involved in regulating calcium and has functional overlap with Phospholipase C. However, evidence suggests that PLD1 has a more prominent role in neurons as it is localized to synapses, 61 involved in BDNF signaling (a key dopaminergic neurotrophic factor 62 ) regulating neurotransmitter release, 61 and is involved with neuronal development. 63 PLD1 is, therefore, a central component linking neuronal physiological calcium signaling with synaptic activity. The PLD1 protein has previously been implicated in PD pathogenesis.
In cell lines, PLD1 enzymatic activity is depressed by the hallmark PD associate protein, a-synuclein. 64 However, a-synuclein clearance was positively regulated by PLD1 activity. 65 Although seemingly contradictory, these data do support the premise for a role of PLD1 in -PD pathophysiological mechanism.
Our data demonstrate that GBA-N370S iPSC-dopamine neurons also have significantly reduced levels of iPLA2. iPLA2 is encoded by the PARK14 gene and pathogenic mutations in the gene decrease calcium influx into intracellular stores, hindering calcium replenishment. 47 Reduced iPLA2 expression in iScience Article GBA-N370S iPSC-dopamine neurons suggests that calcium store replenishment is diminished, consistent with our findings that calcium signaling is strongly perturbed in GBA-PD. Our data show mitochondrial calcium signaling deficits revealed by either ionomycin or CCCP treatment. We propose that this deficit may be because of perturbed calcium release mechanisms, or a reduction in organelle content. The reduction in PLD1 and iPLA2 expression indicates that organelle calcium release and content are both impaired in GBA-N370S cellular phenotypes, although the exact mechanism by which the GCase mutation drives these changes in calcium biology is currently unclear.
Previous work by Schö ndorf et al. 6 in GBA-L444P iPSC-dopamine neurons showed an increased release of calcium from the ER. 6 There are a number of methodological differences between their study and ours which mean the results cannot be directly compared. For example, we used treatment with CPA, a SERCA pump inhibitor, to measure ER calcium leak, whereas Schö ndorf et al. used caffeine, a RyR2 agonist which induces acute organelle calcium-dependent release. No changes in ER stress proteins, nor of calreticulin levels, were reported by Schö ndorf et al. 6 Despite these methodological and phenotypic differences, as well as the different GBA mutations studied, the implication is clear that calcium dysregulation is a central pathophysiological aspect of GBA-PD.
Mitochondrial dysfunction is well established in genetic and sporadic PD 66 and our work adds detailed new mechanistic information. In our GBA-PD model, mitochondrial function is impaired from an early stage of neuronal maturity and, as age is the major risk factor for PD, may contribute to the death of vulnerable dopaminergic neurons in the aging brain. Cell death in GBA-PD driven by dysfunctional mitochondria has been recently proposed to result from loss of GCase function. Both mitochondrial calcium release and mitochondrial membrane potential were decreased in a conditional knock-out Gba mouse model of Gaucher's disease, 67 a lysosomal storage disorder caused by homozygous GCase mutations, resulting in a reduced ATP:ADP ratio. Here, we confirm mitochondrial calcium dysregulation and then demonstrate a bioenergetic deficit in ATP production through reduced OCR in GBA-PD, likely driven by the decrease in Dcm. Furthermore, we also show the effect of energetic failure on the electrophysiological activity and synaptic function of the dopamine neurons vulnerable in PD.
The reduction in mitochondrial calcium which underlies these phenotypes may be caused by reduced calcium flux between the ER and the mitochondria. The ER is a major source supplying calcium to mitochondria after IP3R3 receptor stimulation and subsequent voltage dependent anion channel (VDAC) calcium uptake at ER-mitochondrial associated membranes (MAMs). 68 We propose that reduced calcium flux between these two organelles is as a result of decreased MAM formation because of decreased proximity of ER and mitochondria in GBA-N370S iPSC-dopamine neurons. This is consistent with the disruption of ER-MAM function being a common pathological mechanism in PD.
Recent work has shown that synaptotagmin-1 is important for spontaneous release of neurotransmitter in general 51 and specifically modulates spontaneous rather than activity-driven dopamine release from neurons. 69 Given that we find changes specifically in spontaneous rather than evoked activity in GBA-N370S iPSC-dopamine neurons, we investigated synaptotagmin-1 expression in our model. We found a significant decrease in synaptotagmin-1 in GBA-N370S iPSC-dopamine neurons compared to controls in whole cell lysates, although we did not measure synaptotagmin 1 levels at the synapse or on synaptic vesicles. Synaptotagmin-1 drives neurotransmitter vesicle release as presynaptic calcium levels rise. Reduced levels of synaptotagmin-1 would result in less efficient neurotransmission which would be compounded by reduced calcium signaling. Synaptotagmin-1 therefore represents a possible mechanism by which pathologically depleted neuronal calcium signaling may manifest as aberrant synaptic activity, which might be tested by measuring synaptotagmin 1 levels at the synapse or on synaptic vesicles. Changes in synaptotagmin-1 do not rule out a possible role for other pre-synaptic proteins, nor does a lack of alteration to K v or Na v channel current rule out a role for other channels (BK, HCN etc) in the phenotypes observed.
The work we present utilizes the powerful platform of human iPSC-derived neuronal cultures, providing a human model system relevant to human neurodegeneration. However, the data presented here are from an in vitro monoculture cell model lacking the structural and multi-cellular complexity of a human brain. For this reason, any compensatory mechanisms likely induced by the aberrant phenotypes described may be missed. Further investigation in more complex co-cultures, organoids or animal models are vital in the future to fully understand the scope of the disease. iScience Article Overall, we report the first description of synaptic dysfunction in a GBA-PD model. Although other PD models exhibit altered dopamine content or release, these phenotypes need not reflect dysfunction at the synaptic level as alterations in neurotransmission may be because of changes in dopamine degradation or reuptake. Here, we report aberrant calcium homeostasis which may contribute to synaptic changes through a mechanism of depleted calcium and energy production because of disrupted mitochondrial function, and compromised bioenergetics, consistent with previous work. This occurs in conjunction with reduced levels of the synaptic vesicular protein, synaptotagmin-1, leading to a low efficiency of neurotransmitter release in response to rising levels of pre-synaptic calcium. Taken together, these data represent changes which may have a cumulative contributory effect to chronically reduce neuronal function, and lead to the late-stage neuronal susceptibility and subsequent degeneration of dopamine neurons in PD. The mechanisms described here represent, early pathological alterations which could potentially be targeted in GBA-PD patients to provide an early intervention before neuronal death.

LIMITATIONS OF THE STUDY
Although we found that critical players in calcium signaling were reduced in expression, we limited our work to looking at proteins either with a specific neuronal role (PLD1) or previously implicated in PD (iPLA2), and so our analysis is therefore by no means exhaustive. Other proteins may also contribute to the calcium dyshomeostasis and may represent viable targets. In addition, although the combination of approaches used focused on providing evidence for deficits in mitochondrial calcium stores, other cellular stores and organelles, such as lysosomes, may also be affected. It is expected that such changes would also impact disease relevant pathways.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Generation of iPSC derived dopaminergic neurons
Neuronal differentiations 7 were performed using induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts obtained from donors of the Oxford Discovery Cohort (Table of iPSC  Cells were then dissociated with StemPro Accutase (Life Technologies) and re-plated onto Geltrex at 600,000 cells/cm 2 for coverslips and half area 96 well plates, and 300,000 cells/cm 2 in 6 well plate. Cultures were treated at day 22 with 1 mg/mL mitomycin C in NB medium for 1 h to remove proliferating cells and washed with neurobasal medium before returning to fresh maturation medium. Media was subsequently changed every 3-4 days for the remaining period of maturation up to the respective timepoints of 35 and 70+ DIV. iPSC neuronal cultures were maintained at 37 C, 5% CO 2 . Cells were shown to be TH positive and differentiate similarly between control and GBA mutation lines ( Figure S1). Electrophysiological characterisation illustrated large A-type K v channel current, as well as a hyperpolarising sag in current clamp mode indicative of functional HCN channel expression (Figure S2). Both of these are indicative of nigral neurons. Finally, regular and spontaneous action potential persisted in the presence of network activity inhibition providing evidence of pacemaker activity, hallmark to SNpc neurons.

Preparation of cell extracts
Cells were extracted with cold PBS and centrifuged at 500 3 g for 5 min. Cell pellets were snap frozen and stored at À80 C until needed. Frozen cell pellets were lysed in 150 mL RIPA buffer (TRIS 1 M PH8, iGEPAL, Sodium Deoxy 10%, protease inhibitor cocktail (Sigma, 1 tablet/10 mL), phosSTOP (Sigma, 1 tablet/10 mL)) and briefly sonicated on ice (15 Amp, 10 s). The lysates were then centrifuged 20 min at 4 C at full speed. Protein concentrations were estimated using the Pierce BCA protein assay kit (ThermoFisher) following the manufacturer's instructions. The standard curve was built using a Bovine serum albumin standard. The absorbance was read on the Pherastar (BMG Labtech) at 562 nm.

SDS-PAGE and western blots
Extracted samples were diluted in homemade 6x Laemmli Buffer (for 10 mL: 20% SDS, 30% b-mercaptoethanol, 60% glycerol, 0.012% Bromophenol Blue, 375 mM Tris pH 6.8) and boiled at 90 C for 5 min (the exception were samples for PLD1 assessment which were left unboiled). Samples were loaded on a 4-15% gradient gel (Bio-Rad) and separated via SDS-PAGE (1 h, 120 mV) using a homemade running buffer (144 g glycine, 30.3 g TRIS Base, 10 g SDS for 1 L of 10X buffer). Proteins were then transferred onto a midi PVDF membrane using the High Molecular Weight protocol on the Trans-Blot Turbo Transfer System (Bio-Rad).
The membrane was blocked in TBS-Tween0.1%-Milk 5% for 1 h at room temperature and then incubated overnight at 4 C with the primary antibody in TBS-Tween0.1%-Milk 1%. For the scope of this paper, membranes were separately probed with the following antibodies: PLD1 (Cell Signaling, 3832S), iPLA2 (Merck Millipore, 07-169-I), Synaptotagmin-1 (Cell Signaling, 3347S). Each membrane was then washed 3*10 min in TBS-Tween 0.1% and incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (Bio-Rad) in TBS-Tween0.1%-Milk 1%. Finally, the blot was washed and imaged on the ChemiDoc-Touch (Bio-Rad) with ECL substrate (GE Healthcare). After a rapid wash in TBS-Tween 0.1%, the membrane was stained with an HRP-conjugated antibody directed toward b-actin (Bio-Rad) and imaged.
Densiometric analysis of the protein bands was performed using the volume analysis tool on ImageJ. Each measurement was normalised to actin prior to the comparison of protein abundance between control and GBA-N370S iPSC-dopamine neurons. Due to differentiation variability, controls were included in every ''batch differentiation'' and GBA ratio was normalised to this where control was equal to 1.

Lentiviral transduction for PLD1 knockdown
24 h before transduction, 2.5 x 10 5 SH-SY5Y cells were seeded in 2 mL of complete medium into 6-well plates. Virus was titrated and the volume of virus used was calculated using an MOI of 0.7. Viral vectors were used to transduce cells in the presence of polybrene (8 mg/mL, Millipore). A full media change was performed 24 h post-transduction. Puromycin selection (1 mg/mL, Millipore) was started 24 h post viral transduction, and then gradually increased to 2 mg/mL for the following cell passages. Cas9-GFP expression was induced with Doxycycline (1 mg/mL) 48 h post transduction and was confirmed with fluorescence microscopy (EVOS). Knock out of PLD1 was confirmed using qPCR with the following primer set (5 0 -CCTGCTTTCTTGATGTTCTTTGC-3 0 , 5 0 -GACTGCCTTGACAGGCTTAGA-3 0 ).

Measurement of mitochondrial respiration
Oxygen consumption rate (OCR) was measured using an XF96e Extracellular Flux Analyzer (Seahorse Bioscience). Cells were seeded on XF96-well cell plates on day 20 and further matured until day 35 or 70 before analysis. On the day of the assay, medium was replaced with XF Base Medium (Seahorse Bioscience) supplemented with 10 mM Glucose (Sigma-Aldrich-Aldrich), 1 mM Sodium Pyruvate (Sigma-Aldrich-Aldrich) and 2 mM L-Glutamine (Thermo Fisher Scientific). Cells were then incubated at 37 C in a non-CO 2 incubator for 1 h. Changes in oxygen consumption were measured following sequential injection of the ATP synthase inhibitor oligomycin (1 mM; Sigma-Aldrich), the mitochondrial uncoupler p-triflouromethoxyphenylhydrazone (FCCP 1 mM; Sigma-Aldrich), and the Complex I and III inhibitors Rotenone and Antimycin A (0.5 mM; Sigma-Aldrich). Values were normalized to total protein content of each well and analyzed according to the manufacturer's guidelines.

Proximity ligation assay
To measure ER-mitochondrial proximity, 75 cells were fixed with 4% PFA and stained for TH (1:500 Millipore). Cells were then blocked for PLA and incubated with primary antibodies against IP3R3 (1:500 Merck) and VDAC1 (1:500 Abcam) ON. After washing PLA probes anti-mouse and anti-rabbit were used to complete the PLA reaction as per manufacturer's instructions (Merck Sigma). DAPI was used as a nuclear stain and the puncta from 25 neurons per line and differentiation were counted blindly to determine average puncta per TH neuron.

Statistical analysis
For the analysis of Seahorse assay and JC 10 assays we identified the difference between Conditions A and B, using linear mixed modeling. Because the data of both conditions (A and B) were drawn from different differentiations, we assumed 'differentiation' as the random effect in our model, whereas the 'condition' was represented as the fixed effect. Assuming the effect of differentiation on the response variable (Data) same in every group (i.e. random intercepts model), the model was formulated as M0: Data $ Condition + (1 | Differentiation). We further assumed the differentiation to have a different effect for each group and formulated our random slopes model as M1: Data $ Condition + (1 + Condition | Differentiation). We compared both models using the likelihood ratio test (LRT), the Akaike information criterion (AIC), and the Bayesian information criterion (BIC). Since the random slopes model was not providing significant improvement over random intercepts model in terms of LRT, AIC, and BIC, we preferred to continue our analysis with the random intercepts model (M0). Data were analyzed with the use of R statistical software version 4.0.0. Single cell electrophysiological analysis was performed using Prism6 software (Graphpad, Inc.). Direct comparisons were made by Student's t-test (two-tailed, herein, t-test). Western blot analysis was performed using Mann-Whitney U Test. All significances are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All of the statistical details of experiments can be found in the Figure